In Soybean Cultures.
Two rusts are reported in soybean cultures. The first called American is caused by Phakopsora meibomiae Arthur and the second called Asian rust (ASR) caused by Phakopsora pachyrhizi Sydow.
The fungus that causes soybean rust belongs to the class of basidiomycetes. The species is called Phakopsora pachyrhizi Sydow.
In the specific case of Brazil, the rust was detected in the years 2001/02 and in the very next crop began their control with fungicides. At this time, fungicides “triazoles” alone were applied over a large area with many applications in the soybean production cycle. In the 2005/06 crop some producers (notably in the Brazilian Midwest) complained for the first time, of the rust control failure with the use of these fungicides (mainly DMI). Research conducted in experimental field and laboratory confirmed the reduced sensitivity (resistance) of fungal strains to triazole fungicides.
In the 2013/14 crop were cultivated 29.4 million hectares, with a yield of 3.0 t/ha, with a total production of 82.6 million tons of soybeans.
According to a survey conducted by Kleffmann (2014), in this last soybean crop, on average, three applications/ha were made for the control of P. pachyrhizi (Pp). However, in regions where the second crop of soybeans is cultivated up to 11 applications/ha were made in the same area. This practice speeds up the selection of Pp mutants with resistance to fungicides.
The extension of the treated area, the isolated use of fungicides site specific and the large number of applications in the same crop, faced with anti-resistance strategies recommended by the FRAC (2012) to prevent or delay the emergence of resistant strains, show early occurrence of the risk of Pp resistance to fungicides in Brazil.
It is known from the prior art that fungicides used in soybean and wheat crops fall into two chemical groups with site specific mechanism of action, which are: triazoles, or demethylation inhibitors (DMIs)—included therein the tebuconazole, which for a long time was very efficient; and strobilurins, or outside quinone inhibitors (QoIs). More recently a third chemical group has been used, namely the enzyme succinate dehydrogenase inhibitors (SDHI). The combination of the DMIs with QoIs has been used for more than 12 crops for the control of ASR. Additionally, reducing the sensitivity of Pp to DMI burdened fungicidal action of QoIs in the mixtures. From the 2012/13 crop it was noted that Pp sensitivity was reduced to mixtures of DMIs+QoIs and QoIs+SDHI). More than 50% of soybean areas in Brazil receive a second annual crop, which is called “safrinha” (when it is corn/cotton) or “winter crop” (in the southern regions, with winter crops for wheat, oats, canola, rye etc.). This fact causes various populations of fungi to e also be selected, and causes them to become increasingly resistant to fungicides and mixtures above referenced.
In Wheat.
The fungus that causes the yellow leaf spot of wheat belongs to the class of Imperfect Fungi and is called Drechsiera tritici-repentis (Died) Shoemaker (Dtr) in anamorphic form and Pyrenophora tritici-repentis (Died.) Drechsler in teleomorphic. The yellow leaf spot of wheat was recorded in Australia (Rees & Platz, 1979), Canada (Wright & Sutton, 1990) and the United States (Hosford, 1981). In South America, Dubin (1983) found it in Colombia, Ecuador and Peru. In Argentina it was first observed to the north of Buenos Aires Province in the 1980s (Annone, 1985). In Brazil, the first reference about this disease was made by Costa Neto (1967), having the same being detected in Rio Grande do Sul, in 1959, in the city of Dom Pedrito. Later, Mehta (1975) reported the occurrence of an epidemic of the disease in the state of Parana.
In Brazil, until 1976, it was not allowed the use of fungicides in wheat aerial organs. A milestone for its use was the UNDP/FAO Agreement with the Experimental Station of Passo Fundo, of the Ministry of Agriculture—today Embrapa Wheat.
In wheat crops, on average, three applications per ha/crop are usually made.
According to prior art information, the first attempts to apply fungicides in the aerial part of the wheat crop was around the year 1958 by researchers from the Federal University of Pelotas (today Embrapa Clima Temperado), to control the disease giberela.
At the time, the South-Brazilian Commission of Wheat Research recommended applications of fungicide at booting and flowering, which was used for a decade (between 1976 and 1986), until the recommendation of triadimefom.
In the wheat crop of 2003 technical assistants and producers complained for the first time, about the failure to control leaf spots after continuous use for 20 years of DMI+QoI fungicides.
The area cultivated with wheat crop in 2012/14 reached 2.2 million ha. Control of the disease known as “tan spot of wheat” was made with the use of mixtures, but with very poor (<50%) and inefficient response, due to the development of resistance. Therefore, companies and research institutions are committed to developing an anti-resistance strategy, so that the said disease control efficiency is back to the 80% to 90% level.
Sensitivity Reduction (SR) of Fungi to Fungicides.
It is known that sooner or later, during the years of commercial use of a fungicide, may arise a mutant individual in the target pathogen population that is not sensitive enough to be controlled satisfactorily. It multiplies increasing its population silently.
Generally, the SR is in response to the repeated use of a fungicide with the same mechanism of action, in a large area with many applications in the crop cycle. The first evidence of this change is observed by the producer, that complains about “control failure.” In this situation the control went from “efficient and economic” to “inefficient and uneconomical.”
With respect to the term “sensitivity reduction”, the term “reduction” should be used, preferentially, instead of the term “loss of sensitivity.” SR is proven in the lab when there is an increase in sensitivity reduction factor, i.e., SRF (>1).
The term is used to previously sensitive fungal strains, which, through variation mechanism such as mutation, significantly reduced their sensitivity to fungicide (SRF>1.0).
The science of fungicides describes the resistance of a fungus to a fungicide with site specific mechanism of action (for example, DMI, QoI and SDHI) can be cross or multiple. Cross-resistance occurs within the same group, as for triazoles (cyproconazole, epoxiconazole and tebuconazole), and also for strobilurins (azoxystrobin, picoxistrobia, pyraclostrobin and trifloxystrobin). However, it is worth noting the occurrence of multiple resistance when the same strain of the fungus has a reduced sensitivity both with respect to triazoles as with respect to strobilurins.
With regard to soybean, particularly with soybean rust, it is likely that both the cross-resistance and the multiple are occurring; i.e., resistance to all triazoles and all strobilurins. And in wheat, it was proved that only cross-resistance is being checked.
The situation proved, on this account, to be worrying. Faced with this fact, companies and institutions began to wonder what could be done to rescue the control levels (between 80% and 90%) of triazoles and strobilurins, isolated or in mixture.
Reduction of Chemical Control Efficiency
a. Soybean Rust—Demethylation Inhibitors (DMIs)-Fungicides
As occurred with flutriafol, tebuconazole has become widely used and with high efficiency, being the reference fungicide in controlling rust, but not for long.
In order to clarify the facts, experiments conducted at the Foundation MT, Rondonopolis, by the University of Rio Verde and institutions participating in the Cooperative Tests of Fungicides (beginning in the 2003/04 crop), confirmed the reduction of control efficiency. It was proved the reduction of control effectiveness by comparing the performance of DMIs in the 2005/06 crop with the (2012/13) crop in results of research conducted at the University of Rio Verde. In the 2005/06 crop, the average rust control by DMIs was 90.3. After eight years, corresponding to the 2012/13 crop, the control of DMIs was 52.0 with a reduction in efficacy of 42% (Table 1). (problem)
TABLE 1Reduced soybean rust control by DMIs fungicides appliedpreventively in control (%) and control reductionCropsReductionFungicide2005/062012/13(%)Cyproconazole96.052.045.9Epoxiconazole80.040.050.0Tebuconazole95.064.032.8Average90.352.042.0Source: Silva et al., 2013.
The reduction of the sensitivity of Pp to tebuconazole and cyproconazole fungicides, controlling only 42 and 38%, respectively, was also demonstrated by Godoy and Palaver (2011). At this time, the mixture still showed no reduction in efficiency; cyproconazole+azoxystrobin, 72% and epoxiconazole+pyraclostrobin, 88% control with an average of the mixtures of 80% of control. Probably, at this time, the efficiency was ensured by QoIs as the average of the DMIs was only 40% (Table 2).
TABLE 2Control reduction of soybean rust severity, evaluatedby the area under the disease progress curve (AUDPC)by some fungicides in crop 2010/11SeverityControlTreatments(%)(%)Control74.0 a—Tebuconazole49.9 b42Cyproconazole58.1 b38Cyproconazole + o azoxystrobin14.8 c72Epoxiconazole + pyraclostrobin9.0 88Source: Modified Data of Godoy and Palaver (2011).
An example that reinforces the reported fact is the gradual reduction of tebuconazole control over the crops using it covering the period beginning in 2004/05 to 2013/14 (as FIG. 1).
From the 2003/04 crop, soybean rust control efficiency (ASR) by tebuconazole, was reduced by 7.2% per year (see also FIG. 1).
Therefore, the fact is that today there is a problem that lies in the fact that with this reduction in speed in two more crops, it is likely that soybean rust control by tebuconazole reaches zero. So again companies are faced with the problem of how producers can take precautions to ensure ASR control over 80%? And yet, what would be the amount of damage from lack of anti-resistance strategy? (problems)
Soybean Rust—Qols Fungicides
The reduction of control by the mixtures can also be attributed to sensitivity reduction of Pp to QoIs. From the 2008/09 crop was detected early reduction in the control of azoxystrobin and reached only 16% of control in the 2013/14 crop (see FIG. 6).
Using the equation y=−13.8x+92.8, it was determined that from 2009/10 the efficacy of this fungicide has been reduced by 13.8% per year and ending by reaching in the 2013/14 crop, only 16% of control (see FIG. 6).
Comparing the reduction of tebuconazole control with the azoxystrobin, the current level of control is similarly low. However, the difference is in the shortest time required by DMI and higher with QoI to achieve this same level “low”
Soybean Rust—Fungicide Mixtures Composed of DMI+QoI
What has been checked with the latest crops is the reduction of rust control by mixtures of triazoles+strobilurins. Questioned whether the low control presented by mixtures can be attributed to greater reduction of the sensitivity of the fungus to DMIs. Or, if the lower control of the mixtures can be attributed to reduced sensitivity of Pp to QoIs.
The results of cooperative tests of fungicides, coordinated by Embrapa Soybean, Londrina, can bring the answer to these questions. The following graphs of FIGS. 2, 3, 4 and 5 show the reduction of rust control by fungicide mixtures traditionally used in soybeans over the past crops.
An example with cyproconazole+azoxystrobin mixture is shown in FIG. 2. This mixture has been used since 2003/04, when it exhibited an efficiency of 90%.
Using the equation y=−6.5429x+90.067, you can calculate that from the 2007/08 crop the effectiveness of this mixture has been reduced by 6.54% per year, reaching in the 2013/14 crop only 41% of control (see FIG. 2).
Using the equation y=−9.0x+100.0, one can calculate that from 2008/09 the effectiveness of the mixture cyproconazole+azoxystrobin has been reduced by 9.0% per year, reaching 37% control in the 2013/14 crop (see FIG. 3).
The mixture “epoxiconazole+pyraclostrobin” has been used since 2007/08. With the equation y=−12.6x+99.0 [where y=control (%) and x=the percentage of annual reduction of control], you can calculate that from the 2009/10 crop the effectiveness of this mixture has been decreased by 12.6% per year, reaching in the last crop 23% (see FIG. 4).
According to the equation y=−9.0x+100.0, from the 2008/09 crop the average effectiveness of mixtures “cyproconazole+azoxystrobin”, “cyproconazole+picoxystrobin” and “epoxiconazole+pyraclostrobin” over six crops has been reduced by 9.0% per year, reaching 37% in the last crop (see FIG. 5).
In Wheat
In the 2003 wheat crop for the first time, there was complaint of leaf spot control failure after continuous use of fungicides DMI+QoI, for 20 years. Until that time there were few reports of the sensitivity of the fungus that is causal agent of yellow spot to fungicides (Stolte, 2006; Tonin, 2009; Beard et al, 2009; Patel et al, 2012). The reference concentrations of sensitivity of Dtr to fungicides found in the literature were 0.17 mg/L in average for the epoxiconazole, propiconazole and tebuconazole. Hunger & Brown, determined an IC50 of 0.04 mg/L for propiconazole and 0.19 for tebuconazole; Beard et al, determined IC50 of 0.19 mg/L for epoxiconazole and 0.25 mg/L for tebuconazole). Comparing these values with the sensitivity of isolates of Brazil proves the reduced sensitivity occurring here (see Table 3).
TABLE 3Concentrations for 50% inhibition of mycelium growth (IC50) of fiveisolated from Drechsiera siccans for five fungicides DMIsIsolated (IC50 mg/L)Fungicide01/F3002/RZ03/SF04/F5205/VQAverageCyproconazole0.360.470.320.440.290.37 bEpoxiconazole0.280.280.400.320.400.33 bPropiconazole0.320.510.300.300.300.34 bProthioconazole0.22<0.10.260.250.140.21 cTebuconazole0.660.490.650.610.440.57 aAverage0.360.370.380.380.31CV (%)9.05
Averages followed by the same letter do not differ by 5% of the Tukey test. Average of two experiments.
However, the highest reduction of sensitivity of the Dtr to fungicides and what better explains the control failure claimed by producers is the CI50 obtained for QoIs fungicides (see Table 4).
TABLE 4Inhibitory Concentration of 50% of the spore germination (CI50) of fungicides forfive isolates of Drechsiera tritici-repentis to strobilurin fungicidesIsolated (CI50 mg/L)Fungicide01/QTZ02/ONX03/HZT04/GUA05/CDAverageAzoxystrobinA > 40 aA > 40 aA > 40 aA > 40 aA > 40 aA > 40 aKresoxim methylA > 40 aA > 40 aA > 40 aA > 40 aA > 40 a>40 aPicoxystrobinA > 40 aA > 40 aA > 40 aA > 40 aA > 40 a>40 aPyraclostrobinD 0.75 bB 0.85 bC 0.78 bE 0.58 bA 1.03 b0.80 b TrifloxystrobinA > 40 aA > 40 aA > 40 aA > 40 aA > 40 a>40 aAverage C > 32.15 B > 32.17 C > 32.15 D > 32.11 A > 32.20CV (%)0.02
Averages followed by the same letter do not differ there between by 5% of the Tukey test. Lowercase compare average in the column and capital letters in the line. Average of two experiments.
Taking as Cl50 standard of sensitivity, 0.75 mg/L (Table 4) obtained for pyraclostrobin, it is calculated the reduction factor of the sensitivity of the Dtr to QoIs fungicides (see Table 5).
TABLE 5Reduction factor of the sensitivity of Drechsleratritici- repentis to QoIs fungicides.IsolatedFungicide01/QTZ02/ONX03/HZT04/GUA05/CDAverageAzoxystrobinA > 53 aA > 53 aA > 53 aA > 53 aA > 53 a>53 aKresoxim methylA > 53 aA > 53 aA > 53 aA > 53 aA > 53 a>53 aPicoxystrobinA > 53 aA > 53 aA > 53 aA > 53 aA > 53 a>53 aPyraclostrobinD 1.0 bB 1.13 bC 1.04 bE 0.77 bA 1.37 b0.1.1 b TrifloxystrobinA > 53 aA > 53 aA > 53 aA > 53 aA > 53 a>53 aCV (%)0.02
Cl50 of sensitivity reference of 0.75 mg/L.
When the SRF is close to 1.0 there is no reduction of sensitivity. However, if >1 with different magnitude, it indicates reduced sensitivity of fungi to fungicides. In this case SRF ranged from 1.04 to >53 (see Table 5).
Regarding the state of the art related to patent documents, literature is very broad and comprehensive, although no document reported on the solution to the technical problem, as will be described herein. Just as an example, it stands out:
The aforementioned examples related to soybean and wheat have been so far the most studied due to pathogens Pp and Dt provide amplitude of damage above 50% in their respective crops. However, the concept of resistance should also be extended to all crops including cotton, corn, beans, among others.
Regarding the state of the art related to patent documents, literature is very broad and comprehensive, although no document reported on the solution to the technical problem, as will be described herein. Just as an example, it stands out: International patent application WO 2012040804 A2, entitled: “Synergistic combinations of triazoles, strobilurins and benzimidazoles, uses, formulations, production processes and applications using the same”, which describes an agrochemically synergistic formulation of triazoles, strobilurin and benzimidazoles, in specific proportions to control and/or combat pests and diseases in crops. Also described are their preparation process, use and method of use, as well as the use of triazoles, strobilurin and benzimidazoles in the preparation of a synergistic agrochemically formulation of the invention.
European patent application EP 2719280 A1, titled “Use of N-phenylethylpyrazole carboxamide derivatives or salts thereof for resistance management of phytopathogenic fungi,” which refers to the use of derivatives of carboxamide pyrazole-ethyl-N-phenyl, in particular 3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxylic acid [2-(2,4-dichlorophenyl)-2-methoxy-1-methylethyl] amide for resistance management of phytopathogenic fungi on crops and describes a method for resistance management of phytopathogenic fungi in various crops.
In view of all the foregoing, in order to recover the control levels of some fungicides, the present invention developed a method for preventing/delaying the development of fungal resistance in some crops such as soybeans, wheat, cotton, corn and beans, more specifically to the fungus that is causal agent of soybean rust and the fungus that is causal agent to yellow leaf spot of wheat.